Antibiotic bl869 {62 {11 and method of preparation

ABSTRACT

This disclosure describes a new antibiotic, designated BL869 Beta , produced in a microbiological fermentation under controlled conditions using a new strain of an undetermined species of the genus Streptomyces.

TJnitd States Patent Ellestad et al.

[451 Dec.24,1974

ANTrnto'rtc BL869 3 AND METHOD or PREPARATION Inventors: George Alfred Ellestad, Pearl River;

John Henry Edward James Martin, New City, both of N.Y.; John Norman Porter, Ramsey, NJ.

Assignee: American Cyanamid Company,

Stamford, Conn.

Filed: Sept. 17, 1973 Appl. No.: 398,283

US. Cl. 424/116, 195/80 llnt. Cl A61k 21/00 Field of Search 424/116; 195/80 [56] References Cited UNITED STATES PATENTS 3,678,159 7/1972 Niida et al, 424/116 Primary Examiner-Jerome D. Goldberg Attorney, Agent, or Firm-Edward A. Conroy, Jr.

[57] ABSTRACT 3 Claims, 2 Drawing Figures BRIEF SUMMARY OF THE INVENTION America, Chicago, Illinois (1948). Descriptive details are recorded in Tables I-IV below. Amount of Growth:

Moderate to good on most media; light on Czapeks 5 solution agar. This invention relates to a new antibiotic designated Aerial Mycehufni B13693 and to its production by fermentation, to Aerial mycehum whitish to grayish-white when presmethods for its recovery and concentration from crude em and only trace amountssolutions, and to processes for its purification. The Soluble Pigments: present invention includes within its scope the antibisohlble P g y w h own to brownish; heavy otic in dilute form, as a crude concentrate, and in pure Hlckey and h Kusters Oatflaket Tomato crystalline form. The effects of the new antibiotic on paste'oatmel and wemstems agars' specific organisms, together with its chemical and phys- Reverse ical properties, differentiate it from previously de- Yellowlsh to dark brown Shades most scribed antibacterial agents. Miscellaneous Physiological and Chemical Determinations DETAILED DESCRIPTION OF THE INVENTION Complete liquefaction of gelatin in 7 days; nitrates reduced to nitrites in 7 days; complete peptonization The e ahhhlohe Whleh We have designated on purple milk; no melanin pigments produced; NaCl BL369B ef during the Cultivation under tolerance i 10% but 13%;cellwall analysis showed trolled conditions of a new strain of an undetermined L di i i li id present, Carbon Source g ,9 o e ge u t lgto yqe T new antibiotic tion, according to the method of Pridham, TC. and producing strain was isolated from a praire soil sample G tli b, 1),, J urnal of Bacteriology, 56, 107-1 14, collected near Worthington, Minnesota. A viable cul- (1948) as foll G d tfli ti f bi ture of the new microorganism has been deposited with l t e, d-fructose, i-inositol, lactose, d-mannitol, dh C tu e COU CI OII Labora ory, Norther U izamelibiose,d-raffinose,salicin,d trehalose,d-xylose and tion Research and Development Division, United dextrose; no utilization ofd-melezitose, l-rhamnose and States Department of Agriculture, Peoria, Ill., and has sucrose. been added to its permanent collection. It is freely Mi h l available to the public in this depository under its ac- Aerial mycelium sparse, giving rise to an Occasional Cessloh l f F 9; spore chain. Not enough spore chains are produced to TI'IOClCSCllPllOll and dentification of this new mlcro- Characterize the Spore Chain morphology Mycelium Organism melhtalhed he Culture eeheehoh of h sometimes forms thickened fasciculate elements. Lederle Laborator es Division, American Cyanamld Spores elongate to cylindrical 0 4 5 um 1 5 Company; Pearl R'Veri as Culture ,um, Spore surface smooth as determined by transmiswas supplied by Dr. I-LD. Tresner of these laboratories. sion electron microscopy at 20,000X The following 15 a general description of the microorganism Streptomyces sp. NRRL 5750, based on diag- Dlagnosls nostic characteristics observed. Observations were The general lack of aerial growth of culture BL869 made of the cultural, physiological and morphological 40 rhost media f h determination of Such features of the microorganism in accordance with the Sehhal taxohomlc Cmena as Spore colora methods detailed by Shirling, EB. and Gottlieb, 0., lni a m p gy spore i The finding of temational Journal of Systematic Bacteriology 16, dlammopimehc acid 1n the analys1s of cell walls, how- 313 340, (I966) The chemical Composition of the ever places the organlsm in the genus Strepmmyces. Culture was determined by the procedures given by In v1ew of the lack of adequate crlterla for the characchavalier, H.A., Lechevalier, MP. and Gerber, N.N., teriZaltion 0f sll'eplomyces t0 the Species level he Advances in 4 47 72 1971) tempt has been made to make this determination. The underscored descriptive colors and color chip des- Therefore, culture BL869 will be considered an unignations are taken from Jacobsen, E. et al., Color Hardetermined species of Streptomyces until such a mony Manual, 3rd Edition, Container Corporation of diagnosis is feasible.

TABLE I Cultural Characteristics of Streptomyees s p, NRRL 5750 incubation 14 days Temperature 28C.

MEDIUM AMOUNT OF AERIAL MYCELIUM AND/OR SOLUBLE REVERSE REMARKS GROWTH SPORES PIGMENTS COLOR Yeast extract Moderate No serial mycelium or Yellowish brown; Chestnut brown Surface lightly agar spores moderate (4ni) wrinkled Hickey and Good Trace of grayish-white Brownish; heavy Chestnut brown Surface lightly Tresners agar aerial mycelium (4ni) wrinkled Asparagine Moderate Trace of grayish-white Yellowish brown; Chestnut brown Dextrose agar aerial mycelium light (4ni) Bennetts agar Moderate No aerial mycelium Yellowish brown; Chestnut brown Surface lightly or spores light (4ni) wrinkled Inorganic salts- Moderate Trace of grayish-white Yellowish; light Ycllowish starch agar aerial mycelium Kuster's oat- Moderate No aerial mycelium Brownish; heavy Chestnut brown flake agar or spores (4ni) Czapeks Light No aerial mycelium None Whitish Solution agar or spores Potato-dextrose Good Trace of whitish aerial Yellowish brown Chocolate brown Surface lightly agar mycelium moderate (Spo) wrinkled TABLE I Cultural Characteristics of Streptomyccs NRRL 5750 Incubation 14 days Temperature 28C.

MEDIUM AMOUNT OF AERIAL MYCELIUM AND/OR SOLUBLE REVERSE REMARKS GROWTH SPORES PlGMENTS COLOR Tomato Pastc- Good Trace of whitish aerial Brownish; heavy Chocolate brown Surface with Oatmeal agar mycelium (Spo) deep fissures Benedict's agar Good Trace of whitish aerial Brownish; Chestnut brown mycelium moderate (4ni) Wcinstcins Good Trace of whitish aerial Brownish; heavy Deep Brown Surface finely agar mycelium Mahogany (6pl) wrinkled Pablum agar Very good Trace of whitish aerial Yellowish brown; Chestnut brown Surface with mycclium moderate (4ni) deep fissures TABLE II Micrornorphology of Streptomyces g2. NRRL 5750 MEDIUM AERIAL MYCELIUM AND/R SPORIFEROUS STRUCIURES SPORE SHAPE SPORE SIZE SPORE SURFACE Aaparagine Aerial uwcelium sparse, giving rise to an Elongate to O. -t-O.5 um Smooth as determined Dextrose agar occasional apore chain. Not enough spore cylindrical x by transmission chain to characterize the spore cha'in morph- 1.0-1.5 um electron microscopy elegy. Mycelium sometimes forms thickened at 20,000X fascicles-like elements.

TABLE III Micellaneous Physiological Reaction of Strentomvces NRRL 5750 Temperature 28C.

INCUBATION PERIOD AMOUNT OF GROWTH PHYSIOLOGICAL'REACTION Gelatin 7 days Good Complete liquefaction Organic Nitrate Broth 7 days Good Reduced nitrates to nitrites Purple Milk 7 days Good Ctxnplete peptonizntion: no curd Peptoue-Iron 2 t- 48 h'ours Good No mclunln plrzmentn nrcriuecd Yeast. extract agar plus 7, l0 and .1. 151) llaCt 7 days Moderate NuCl tolerance f but 131- 1 Cell wall. Analysis L-Diaminopimelic Acid present. l

TABLE IV lt is to be understood that for the production of the new antibiotic, the present invention is not limited to Carbon Source Utilization 50 this particular microorganism or to microorganisms HWH'MWENRRL fully answering the above growth and microscopic Incubation 14a Tcmpmmc characteristics which are given for illustrative purposes. In fact, it is desired and intended to include the CARBON SOURCE UTILIZATION" use of mutants produced from the described microor- Mmbinosc 3 ganisms by various means such as exposure to X- d-Gal c 3 radiation, ultraviolet radiation, nitrogen mustard, ac-

; tinophages, and the like.

Lactose 3 d-Mlmniwl 3 Fermentation Process d-Melczttose 0 2 Cultivation of the microorganism S treptomyces sp. b NRRL 5750 may be carried out in a wide variety of liq- Sali in 3 uid culture media. Media which are useful for the proiffisgi duction of this novel antibiotic include an assimilable Ll-XylQSc 3 source of carbon such as starch, sugar, molasses, glyc- Cumml erol, etc.; an assimilable source of nitrogen such as protein, protein hydrolysate, polypeptides, amino acids, -i omit Utili/ation corn steep liquor, etc.; and inorganic anions and cat- 1 ions, such as potassium, sodium, calcium, sulfate, phos- U i N" Umimm phate, chloride, etc. Trace elements such as boron, mo-

] Poor Utilization lybdenum, copper, etc.; are supplied as impurities of other constituents of the media. Aeration in tanks and bottles is provided by forcing sterile air through or onto the surface of the fermenting medium. Further agitation in tanks is provided by a mechanical impeller. An antifoaming agent, such as lard oil, may be added as needed.

inoculum Preparation Shaker flask inoculum of Streptomyces sp. NRRL 5750 is prepared by inoculating 100 ml. of sterile liquid medium in 500 ml. flasks with scrapings or washings of spores from an agar slant of the culture. The following is an example of a suitable medium:

Molasses 20 gm.

Glucose 10 gm. Beef Extract I gm. Water to I000 ml.

Adjust medium pH to 6.2 with NaOH Tank Fermentation For the production of the antibiotic BL869B in tank fermentors the following medium is preferably used:

Corn steep liquor 25 gm.

lucose 30 gm. (NHQ SO, 3.3 gm. Calcium carbonate 9 gm. Water to 1000 ml.

Each tank is inoculated with 3 to 10% of inoculum made as described above. Aeration is supplied at the rate of 0.5-1 .0 liter of sterile air per liter of broth per minute and the fermenting mixture is agitated by an impeller driven at 200400 r,p,m, The temperature is maintained at 25-29C., usually at 28C. The fermentation is ordinarily continued for 65-90 hours, at which time the mash is harvested.

Isolation of Antibiotic BL869/3 After the fermentation is completed, the fermented mash containing BL869B is filtered. The filtrate is treated with sodium fluoride (0.2%) at pH 6.5, stirred for 1 hour and then filtered. The filtrate is percolated at pH 6.5 through 5 liters of Amberlite IRC-SO (Na contained in a 4 X 36 column with a flow rate of about 270 ml./minute. The column is washed with 30 liters of distilled water and water-wash effluent is discarded. Elution of the adsorbed antibiotic activity is carried out with 72 liters of 0.3N sulfuric acid in aqueous acetone. Active fractions are combined and the pH adjusted to 5.0 with sodium hydroxide. The acetone is removed in a still. The remaining 53 liters are adjusted to pH 6.8. To these 53 liters are added 5.3 liters of ethyl acetate and 530 ml. of bis-(2-ethylhexyl)phsophate followed by pH adjustment to 8.0 with sodium hydroxide. The ethyl acetate phase is separated and treated with 12 liters of distilled water. The pH is adjusted to 2.0 with sulfuric acid. The aqueous phase is separated and adjusted to about pH 5 with solid barium hydroxide. The mixture is filtered and the residual ethyl acetate is removed. The remaining concentrate (about 4 liters) contains BL869B. This concentrate is passed very slowly through an Amberlite XAD-2 column (2 X inches) at neutral pH. The column is washed with water.

The Amberlite XAD-2 column is then eluted with methanol containing 0.2% acetic acid. After concentration, the aqueous residue is freeze dried yielding BL869B of about 20% purity. This product is dissolved in a minimum amount of water and adborbed onto a I /2 X 18 inch column of carboxymethyl cellulose in the sodium form (pH 6 The column is washed with dis tilled water until the eluate is essentially colorless. Elution with 5% sodium chloride provides the activity in a volume of 750 ml. as monitored by UV. at 320 mu. This eluate is adjusted to pH 7 with 5N sodium hydroxide. A 25 ml. portion of bis-(2-ethylhexyl)-phosphate is added and the pH readjusted to 7.2. One liter of ethyl acetate is added and the phases are mixed with the aid of an air stirrer. The organic layer is then separated and stirred with 500 ml. of water and the pH is adjusted to 2.5 with 18N sulfuric acid. The aqueous layer is neutralized to pH 6.5 with solid barium hydroxide. Filtration and freeze drying yield BL869B of about 15% purity. Extraction of this material with methanol by stirring overnight at room temperature yields, after removal of the solvent, solid BL869B of about purity. This BL869,8 is dissolved in a small amount of the lower phase of the system n-butanol-methanol-acetic acid-water l60l2-2I 20) and adsorbed on the top of a 200 gm. Whatman cellulose powder (grade CF 1 1) column equilibrated with the bottom phase of the solvent system. The activity is eluted with the upper phase and the active fractions are combined and treated with a large volume of petroleum ether. The aqueous phase is separated and the residual solvent is removed under vacuum. After freeze drying, the product BL869B appears as an amorphous buffcolored material of high purity which is very soluble in water and to a lesser extent in methanol.

In vitro Activity BL869B is active in vitro against a variety of gram positive and gram negative bacteria when tested by the standard agar dilution streak plate procedure. The results of such a test appear in Table V and are reported In vivo Activity The compound BL869B is active in vivo against a variety of organisms. This new antibacterial is thereby potentially useful as a therapeutic agent in treating bacterial infections in mammals. This new antibacterial can be expected to be usefully employed for treating or controlling bacterial infections by parenteral administration.

The usefulness of this new antibacterial agent is demonstrated by its ability to control systemic lethal infections in mice. BL869B shows in vivo antibacterial activity in mice against Escherichia coli, Proteus mirabilis, Salmonella typhosa, Herellea Species No. 10, Staphylococcus aureus Strain Smith, Stapliylocaccus aural/s Strain Rose, treptococctts pyo g e r g s and Diplococcus pneunzoniae whenadministered by a single 0.5 ml. subcutaneous dose suspended in 0.2% aqueous agar to groups of Carworth Farms CF1 mice, weighing about 20 gm., infected intraperitoneally with a lethal dose of these bacteria in 10, 10*, undiluted, 10' 10, 10, 10' and trypticase soy broth (TSP) dilutions, respectively, of a 5 hour TSP blood culture. Table VI illustrates the in vivo activity of BL869B against these bacteria.

TABLE VI Single subcutaneous Alive/Total Mice Tested 7 Days After Dose mg./kg. Injection Escherichia cali No. 31 l 128 10/10 64 9/10 32 19/20 16 8/10 8 2/10 4 IIIO 2 0/10 Infected non-treated 39/40 Mice died within 2 days after controls infection Proteus mirabilis No. 4671 128 13/20 64 9/20 32 N20 16 0/20 8 0/10 Infected nontreated 40/40 Mice died within 2 days after controls infection Salmonella lyp/wsa 6539 64 8/20 32 10/20 16 /20 8 3/20 4 1/10 Infected non- 37/40 Mice died within 2 days after treated controls infection Herelleu Specie: No. 10

64 13/20 32 8/20 16 0/20 8 0/20 Infected non- 48/50 Mice died within 2 days after treated controls infection Staphylococcus aureus Strain Smith 64 /20 32 20/20 16 7/20 8 0/20 Infected non 38/40 Mice died within 2 days after treated controls infection Staphylococcus aureus Strain Rose 128 18/20 64 10/20 32 H20 16 1/20 Infected non- 36/40 Mice died within 1 day after treated controls infection Slrepmcoccus pyagenes C203 128 lO/IO 64 20/20 32 20/20 16 5/20 8 H20 Infected non- 40/40 Mice died within 2 days after treated controls infection Diplotoccus pneumuniae SVl 128 9/10 64 20/20 32 20/20 16 9/20 8 0/20 4 0/20 Infected non- 40/40 Mice died within 2 days after treated controls infection The invention will be described in detail in conjunction with the following specific examples:

EXAMPLE I Inoculum Preparation Molasses 20 gm. Glucose 10 gm. Beef Extract 10 gm. Water to 1000 ml.

The washed or scraped spores from an agar slant of Streptomyces sp. NRRL 5750 were used to inoculate two 500 ml. flasks each containing ml. ofthe above sterile medium, which had been adjusted to pH 6.2 with sodium hydroxide. The flasks were placed on a rotary shaker and agitated vigorously for 48 hours at 28C. The resulting flask inoculum was transferred to a 20 liter glass fermentor containing 12 liters of the same sterile medium. The inoculum mash was aerated with sterile air while growth was carried out for about 48 hours, after which the contents were used to seed a 300 liter tank fermentor.

EXAMPLE 2 Fermentation A fermentation medium was prepared according to the following formula:

Corn steep liquor 25 gm. Glucose 30 gm. Ammonium sulfate 33 gm. Calcium carbonate 9 gm. Water to 1000 ml.

The fermentation medium was sterilized at C. with steam at 20 pounds pressure for 60 minutes. The pH of the medium after sterilization was 6.4. Three hundred liters of sterile medium in a 400 liter tank fermentor was inoculated with 12 liters of inoculum such as described in Example 1. The fermentation was carried out at 28C. using Hodag LG-8 oil as a defoaming agent. Aeration was supplied at the rate of 0.5 liter of sterile air per liter of mash per minute. The mash was agitated by an impeller driven at 250 rpm. At the end of approximately 90 hours of fermentation time the mash was harvested.

EXAMPLE 3 Isolation of BL869B The fermentation broth, prepared as described in Example 2, and having a volume of 265 liters, was treated with sodium fluoride (0.2%) at pH 6.5, stirred for about 1 hour, and then the precipitate was filtered off with the aid of 1% Hyflo Super-cel.

This filtrate was percolated, at pH 6.5, through 5 liters of Amberlite [RC-50 (a methacrylic acid-divinyl benzene ion exchange resin) (Na contained in a 4 X 36 inch column with a flow rate of 270 ml./minute. The column was washed with 30 liters of distilled water and the water-wash effluent was discarded. Elution of the adsorbed antibiotic activity was carried out with 72 liters of 0.3N sulfuric acid in 10% aqueous acetone. Active fractions were combined and the pH adjusted to 5.0 with sodium hydroxide. The acetone was removed in a still. The remaining 53 liters were adjusted to pH 6.8. To this was added 5.3 liters of ethyl acetate and 530 ml. of bis-(2-ethylhexyl)phosphate followed by pH adjustment to 8.0 with sodium hydroxide. The ethyl acetate phase was separated and treated with 12 liters of distilled water and the pH was adjusted to 2.0 with sulfuric acid. The aqueous phase was separated and neutralized to about pH 5 with solid barium hydroxide. The mixture was filtered and the residual ethyl acetate was removed leaving 4 liters of concentrate containing BL869B. This material was passed very slowly through an Amberlite XAD-2 (a polystyrene absorbent) column (2 X inches) at neutral pH and washed with wa ter. The Amberlite XAD2 column was then eluted with methanol containing 0.2% acetic acid. After concentration, the aqueous residue was freeze dried to give 7.5 gm. of crude BL679B (20% purity). The BL869B was dissolved in a minimum volume of water and adsorbed onto a 1 /2 X 18 inch column of carboxymethyl cellulose in the sodium form (pH 6). The column was washed with distilled water until the eluate was essentially colorless. Elution with 5% sodium chloride solutionprovided the activity in a volume of 750 ml. as monitored by ultraviolet at 320 ma. This eluate was adjusted to pH 7 with 5N sodium hydroxide. Twenty-five m1. of bis-(2-ethylhexyl)phosphate was added and the pH readjusted to 7.2. One liter of ethyl acetate was added and the phases were mixed with the aid of an air stirrer. The organic layer was then separated and stirred with 500 ml. of water and the pH was adjusted to 2.5 with l8N sulfuric acid. The aqueous layer was neutralized to pH 6.5 with solid barium hydroxide. This mixture was filtered and the filtrate was freeze dried yielding 4.5 gm. of BL869B of 15% purity. This product was extracted with 200 ml. of methanol by stirring overnight at room temperature. The solvent was removed yielding BL869B of 60% purity.

EXAMPLE 4 Purification of BL869B A 2.6 gm. portion of BL869B of about 60% purity, was dissolved in a small amount of the lower phase of the system n-butanol:methanolzacetic acidzwater (l60:12:2:120) and adsorbed on the top of a 200 gm. Whatman (grade CF 11) cellulose powder column equilibrated with the bottom phase of the solvent system. The activity was eluted with the upper phase and the active fractions were combined and treated with +11.1 (C=0.351 in Ultraviolet Absorption: A,,,,,, 3 235 and 323 mu (e 350 and 640).

A standard infrared absorption spectrum of BL869B hydrochloride prepared in a KBr disc is shown in FIG. 1 of the accompanying drawings. A standard nuclear magnetic resonance spectrum of BL869B hydrochloride prepared in deuterated dimethylsulfoxide is shown in FIG. 2 of the accompanying drawings.

We claim:

1. Antibiotic BL869,8 hydrochloride, a compound which a. is effective in inhibiting the growth of bacteria; and

in its essentially pure crystalline form b. has an optical rotation [01],,

(C=0.351 in methanol);

c. has the following elemental analysis (percent): C,

40.94; H, 5.42; N, 23.54; 0, 11.63; Cl, 16.07;

d. has ultraviolet absorption maxima A F 235 and 323 mp. (y 350 and 640);

e. has a characteristic infrared absorption spectrum as shown in FIG. 1 of the drawings; and

f. has a characteristic proton magnetic resonance spectrum as shown in FIG. 2 of the drawings.

2. A compound as defined in claim 1, antibiotic BL869B hydrochloride, in its essentially pure form.

3. A process for the production of antibiotic BL869B hydrochloride which comprises cultivating Streptomyces sp. NRRL 5750 in an aqueous nutrient medium containing assimilable sources of carbohydrate, nitrogen, and inorganic salts under submerged aerobic conditions until substantial antibacterial activity is imparted to said medium, and then recovering antibiotic BL869B hydrochloride therefrom. 

1. ANTIBIOTIC BL869B HYDROCHLORIDE, A COMPOUND WHICH A IS EFFECTIVE IN INHIBITING THE GROWTH OF BACTERIA, AND IN ITS ESSENTIALLY PURE CRYSTALLINE FORM B. HAS AN OPTICAL ROTATION (A)B25 =+11.1*(C=0.351 IN METHANOL); C. HAS THE FOLLOWING ELEMENTAL ANALYSIS (PERCENT): C, 40.94; H, 5.42; N, 23.54;O,11.63;CL, 16.07; D. HAS ULTRAVIOLET ABSORPTION MAXIMA $MAXCHOH235 AND 323 M$ (Y1CM.1% 350 AND 640); E. HAS A CHARACTERISTIC INFRARED ABSORPTION SPECTRUM AS SHOWN IN FIG. 1 OF THE DRAWINGS AND F. HAS A CHARACTERISTIC PROTON MAGNETIC RESONANCE SPECTRUM AS SHOWN IN FIG. 2 OF THE DRAWINGS.
 2. A compound as defined in claim 1, antibiotic BL869 Beta hydrochloride, in its essentially pure form.
 3. A process for the production of antibiotic BL869 Beta hydrochloride which comprises cultivating Streptomyces sp. NRRL 5750 in an aqueous nutrient medium containing assimilable sources of carbohydrate, nitrogen, and inorganic salts under submerged aerobic conditions until substantial antibacterial activity is imparted to said medium, and then recovering antibiotic BL869 Beta hydrochloride therefrom. 